PROJECT SUMMARY/ABSTRACT Title: Roles of the Fanconi Anemia DNA Repair Pathway in Managing Acetadehyde-induced Replicative Damage and Fork Stability in Esophageal Keratinocytes Esophageal squamous cell carcinoma (ESCC), the sixth deadliest cancer worldwide, has a 5-year survival rate of only 20% and is highly understudied. Treatment options, mainly taxol chemotherapy and/or esophagectomy procedures, are highly invasive and significantly lower the quality of life, thus there is a great need for novel and innovative mechanistic insights in order to develop new therapeutic strategies. One of the major risk factors for ESCC is chronic alcohol consumption, which leads to acetaldehyde build-up. Acetaldehyde (AA), the primary metabolite of alcohol, is a major carcinogen that can interact with DNA to form adducts and interstrand crosslinks (ICLs), which can block the DNA replication machinery and lead to DNA breaks and cell death. ICLs are repaired by the Fanconi anemia (FA) DNA repair pathway, and inactivation of the genes that encode for proteins in the FA pathway results in Fanconi anemia, a genetic disorder that features bone marrow failure and predisposition to malignancies, including leukemia and young-onset squamous cell carcinoma (SCC). Consistently, AA is known to activate the FA pathway, and FA patients are shown to be AA-sensitive. However, the underlying mechanism of how AA activates the FA pathway remains elusive. Our preliminary data shows that acetaldehyde results in a loss of cell viability, replicative DNA damage foci, and Chk1-dependent G2 arrest. We also show that acetaldehyde results in activation of the FA pathway in esophageal keratinocytes and that depletion of the FA pathway exacerbates acetaldehyde-induced cell death. Thus, we hypothesize that acetaldehyde-induced DNA damage results in replicative stress, leading to the activation the FA pathway to preserve replication fork integrity and promote DNA repair. In Aim 1, we will characterize AA-mediated DNA damage response and replication stress in human esophageal keratinocytes. In Aim 2, we will investigate how the FA pathway preserves replicative potential in AA-treated human esophageal keratinocytes. These studies will further our understanding of the mechanisms that drive alcohol-induced DNA damage and esophageal carcinogenesis and help identify more effective therapeutic targets for ESCC treatment and prevention.